Name: Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice (Video) | JoVE Journal
Uploaded: 2012-05-03T13:00:00.000+00:00
Duration: 11 min 16 s
Description: Learn progressive-ratio operant conditioning to measure food reward motivation in mice using high-fat, high-sugar pellets. This method validates how food deprivation and leptin alter motivated behavior.

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5 minutes

This project was supported by grants from the Natural Sciences and Engineering Research Council of Canada (355881) and the Canadian Foundation for Innovation.

*Most important steps.
1. Equipment and Software
<ol>
	<li>Training and testing is carried out in mouse operant chambers equipped with two ultra-light, retractable levers1 positioned on either side of a food receptacle (see Table 1). A stimulus light is located above each lever and there is a house light at the top of the cage. A pellet dispenser is located outside of the chamber. Each chamber is housed inside a sound-attenuating cubicle with ventilating fan. Chambers are connected to a PC computer with MedPC IV software via a "Smart Control" interface cabinet.</li>
</ol>
Note: When ultra-light levers are not available it is recommended that nose-poke be used as the operant response for mouse studies. However, with both options available we suggest selection of ultra-light levers as a more reliable measure of motivation given that nose-poke responses can be triggered by exploratory sniffing.
<ol start="2">
	<li>The MedPC programs used to run operant tasks are written in Medstate notation code which a user can learn with the help of the programming manual provided. Programming provides flexibility for the user to modify existing codes or generate unique experimental parameters. Certain codes can be obtained for free from Med Associates "Medstate Notation Repository" <a href="http://www.mednr.com" target="_blank">http://www.mednr.com</a>. Additional codes may be purchased from Med Associates.</li>
</ol>
2. Mouse Acclimation and Food Restriction
<ol>
	<li>Upon arrival mice are group-housed for at least 10 days to acclimatize to a reverse 12 h light/dark cycle and provided with ad libitum access to standard chow and water. In our case we use adult C57BL/6 mice purchased from Charles River (St. Constant, QC). All mouse manipulations and behavioral experiments are performed during the dark cycle. All procedures involving the use of animals were approved by the CRCHUM Animal Care Committee.</li>
	<li>Mice are subsequently singly-housed and food restricted (70% of their daily food intake) until they reach 90-95% free-feeding body weights in order to facilitate acquisition of lever-press responding. Mice were provided their daily quota of food in the home cage after the termination of session. Upon reaching 5-10% weight loss, the daily food allotted is adjusted to stabilize the lower body weights for the remainder of the training period. Once mice have attained acquisition criteria (see below) they are put back on an ad libitum feeding schedule. Mice regain lost body weight in about 3-4 days.</li>
	<li>Mice are handled in the test room for 3 consecutive days prior to the first training session. The day prior to the start of training 10-15 high-fat and high-sugar (HFHS) pellets are placed into the home cage to prevent the potential influence of food neophobia on operant performance. We use 20 mg HFHS, dust-free, precision food pellets (14 mg also available) containing 48.9% Kcal as fat (Bio Serv, Frenchtown, NJ).</li>
</ol>
3. Operant Training
<ol>
	<li>Mice are initially trained to press the lever on a fixed ratio (FR)-1 reinforcement schedule whereby a single lever press elicits the delivery of a food pellet to the receptacle. Only one lever is designated as "active" (triggering delivery of food reward) and the allocation of right and left levers is counterbalanced between mice. It is important to add at least a 5 second timeout (TO) to the FR1 schedule (FR1/TO-5), during which additional lever-pressing does not result in the delivery of a food pellet. This TO period allows time for mice to consume the food pellet, however longer time outs may be used if the experimenter feels it is necessary for their mouse model. Each FR training session lasts 1 hour or when 50 pellets have been delivered.</li>
	<li>*Acquisition Criteria: The criteria used to determine acquisition of food-maintained operant responding includes: (1) a minimum number of active responses and rewards earned; (2) a measure of discrimination between active and inactive levers, and (3) between-session performance stability. Mice exhibiting discrimination of ≥3:1 for the active versus inactive lever and obtaining ≥ 20 rewards per session over three consecutive sessions are considered to achieve acquisition criteria10. We have observed that the majority (~75%) of C57BL6 mice require about 7-10 days of training to achieve acquisition criteria. Mice that do not reach acquisition criteria by this time undergo further training for an additional 5-7 days. We exclude mice from testing (~5% of cases) when there is no progress by the extra 5th training day. It is important to keep in mind that impaired operant acquisition may be an outcome of certain pharmacological and genetic interventions and should therefore be documented.</li>
	<li>Following three successive sessions of obtaining ≥ 20 pellets, the schedule is increased to FR5/TO-5 seconds in which 5 active lever presses trigger the delivery of the food pellet. Training on the FR5 schedule lasts three days.</li>
	<li>*The mice are then trained in the progressive ratio (PR) schedule of reinforcement. The response ratio schedule during PR testing can be calculated as per Richardson and Roberts (1996)11 using the following formula (rounded to the nearest integer): 
	= [5e (R*0.2)] - 5 
	where R is equal to the number of food rewards already earned plus 1 (i.e., next reinforcer). Thus, the number of responses required to earn a food reward follow the order: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95 and so on. The final ratio completed is the breakpoint.</li>
	<li>A PR session lasts a maximum of 1 hour. Failure to press the lever in any 10 min period results in termination of the session. Performance on the PR schedule of reinforcement is considered stable when the number of rewards earned in a 1 hour session deviates by ≤10% for at least 3 consecutive days.</li>
</ol>
4. Progressive Ratio Testing and Validation
<ol>
	<li>To validate food-maintained operant responding in the PR task, one can assess breakpoint responding following manipulations known to modulate food reward. A relatively easy test to carry out is food deprivation which will increase the motivational state of the mouse for food and thereby increase breakpoints. Mice must first be tested in the PR task during a period of ad libitum feeding until stable, baseline performance is achieved. The day after the last "baseline" day food is removed from the home cage and mice are then tested in the PR task 24 hours after the start of the fast.</li>
	<li>We also investigated whether peripheral administration of the anorectic hormone leptin would have the opposite effect on breakpoint thresholds. Leptin (A.F. Parlow, National Hormone and Peptide Program, NIDDK) was dissolved in sterile PBS. PR testing was carried out following an IP injection of PBS on one day and then following injection of leptin (5 mg/kg) the following day. Leptin was injected one hour prior to PR testing. Breakpoints were compared using a paired t-test (GraphPad Prism).</li>
</ol>
5. Representative Results
When testing the ability of food deprivation to modulate breakpoint responding on a PR schedule of reinforcement, we expect to significantly increase breakpoints as shown in Figure 1. In this experiment a 24 h period of food deprivation produced a ~3.6-fold increase in breakpoints as compared to those obtained in the free-feeding baseline state thereby suggesting that food deprivation increases the rewarding effects of food. In contrast, peripheral administration of the anorectic hormone leptin (5 mg/kg, IP) one hour prior to testing should decrease breakpoint responding as shown in Figure 2.
<img alt="Bar graph comparing baseline vs 24-hour fasting breakpoint data; statistical analysis indicated." src="/files/ftp_upload/3754/3754fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Fasting increases the rewarding effects of food. A 24 h fast significantly increased breakpoints on a progressive ratio (PR) schedule of reinforcement in mice (n=4). Mean±SEM; *p&lt;.05.
<img alt="Bar graph comparing breakpoint levels of Vehicle and Leptin, indicating statistical significance." src="/files/ftp_upload/3754/3754fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Leptin decreases food reward in mice. Peripheral leptin administration (5 mg/kg) decreased PR breakpoint response thresholds in mice (n=4). Mean±SEM; *p&lt;0.05.

<ol><li>Fulton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appetite+and+reward%5BTitle%5D)+AND+%22Front.+Neuroendocrinol%22%5BJournal%5D)">Appetite and reward.</a> Front. Neuroendocrinol. 31, 85-103 (2010).</li>
<li>Zheng, H., Lenard, N. R., Shin, A. C., Berthoud, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appetite+control+and+energy+balance+regulation+in+the+modern+world%3A+reward-driven+brain+overrides+repletion+signals%5BTitle%5D)+AND+%22International+journal+of+Obesity%22%5BJournal%5D)">Appetite control and energy balance regulation in the modern world: reward-driven brain overrides repletion signals.</a> International journal of Obesity. 33, Suppl 2. S8-S13 (2009).</li>
<li>Skinner, B. F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSkinner%2C+BF+%22The+Behavior+of+Organisms%3A+An+Experimental+Analysis%22">The Behavior of Organisms: An Experimental Analysis</a>. , Appelton-Century. (1938).</li>
<li>Hodos, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+ratio+as+a+measure+of+reward+strength%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Progressive ratio as a measure of reward strength.</a> Science. 134, 943-944 (1961).</li>
<li>Hodos, W., Kalman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+increment+size+and+reinforcer+volume+on+progressive+ratio+performance%5BTitle%5D)+AND+%22J.+Exp.+Anal.+Behav%22%5BJournal%5D)">Effects of increment size and reinforcer volume on progressive ratio performance.</a> J. Exp. Anal. Behav. 6, 387-392 (1963).</li>
<li>Stafford, D., LeSage, M. G., Glowa, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive-ratio+schedules+of+drug+delivery+in+the+analysis+of+drug+self-administration%3A+a+review%5BTitle%5D)+AND+%22Psychopharmacology%22%5BJournal%5D)">Progressive-ratio schedules of drug delivery in the analysis of drug self-administration: a review.</a> Psychopharmacology. , 139-169 (1998).</li>
<li>Glass, M. J., O'Hare, E., Cleary, J. P., Billington, C. J., Levine, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+naloxone+on+food-motivated+behavior+in+the+obese+Zucker+rat%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">The effect of naloxone on food-motivated behavior in the obese Zucker rat.</a> Psychopharmacology (Berl). 141, 378-384 (1999).</li>
<li>Brennan, K., Roberts, D. C., Anisman, H., Merali, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Individual+differences+in+sucrose+consumption+in+the+rat%3A+motivational+and+neurochemical+correlates+of+hedonia%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">Individual differences in sucrose consumption in the rat: motivational and neurochemical correlates of hedonia.</a> Psychopharmacology (Berl). 157, 269-276 (2001).</li>
<li>Vaughan, C., Moore, M., Haskell-Luevano, C., Rowland, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Food+motivated+behavior+of+melanocortin-4+receptor+knockout+mice+under+a+progressive+ratio+schedule%5BTitle%5D)+AND+%22Peptides%22%5BJournal%5D)">Food motivated behavior of melanocortin-4 receptor knockout mice under a progressive ratio schedule.</a> Peptides. 27, 2829-2835 (2006).</li>
<li>Haluk, D. M., Wickman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+study+design+variables+and+their+impact+on+food-maintained+operant+responding+in+mice%5BTitle%5D)+AND+%22Behav.+Brain+Res%22%5BJournal%5D)">Evaluation of study design variables and their impact on food-maintained operant responding in mice.</a> Behav. Brain Res. 207, 394-401 (2010).</li>
<li>Richardson, N. R., Roberts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+ratio+schedules+in+drug+self-administration+studies+in+rats%3A+a+method+to+evaluate+reinforcing+efficacy%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy.</a> J. Neurosci. Methods. 66, 1-11 (1996).</li>
<li>Jewett, D. C., Cleary, J., Levine, A. S., Schaal, D. W., Thompson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+neuropeptide+Y%2C+insulin%2C+2-deoxyglucose%2C+and+food+deprivation+on+food-motivated+behavior%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">Effects of neuropeptide Y, insulin, 2-deoxyglucose, and food deprivation on food-motivated behavior.</a> Psychopharmacology (Berl). 120, 267-271 (1995).</li>
<li>Fulton, S., Woodside, B., Shizgal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+brain+reward+circuitry+by+leptin%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Modulation of brain reward circuitry by leptin.</a> Science. 287, 125-128 (2000).</li>
<li>Figlewicz, D. P., Bennett, J. L., Naleid, A. M., Davis, C., Grimm, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraventricular+insulin+and+leptin+decrease+sucrose+self-administration+in+rats%5BTitle%5D)+AND+%22Physiol.+Behav%22%5BJournal%5D)">Intraventricular insulin and leptin decrease sucrose self-administration in rats.</a> Physiol. Behav. 89, 611-616 (2006).</li>
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Production and Free Access of this video-article is sponsored by Med Associates, Inc.

Operant conditioning tasks provide an effective means to evaluate changes in the motivational properties of food. The experimental procedures detailed in the present article permit training mice to lever press for food rewards and stably respond on a PR schedule of reinforcement.
Measuring the amount of food consumed does not provide the same readout as effort-based response measures since not all factors that affect food intake modulate the rewarding effects of food. For example, depleting utilizable glucose by peripheral 2-deoxyglucose administration in rats increases the amount of freely available food consumed but fails to alter break points in a PR task for food12. PR breakpoint responding can also reflect changes in the quality of food rewards. For example, break points are positively correlated with sucrose concentration demonstrating that motivation for sucrose increases in a manner that follows sweet taste8. Here we demonstrate that 24h food deprivation substantially increases PR breakpoints for HFHS food in mice. In contrast, we found that peripheral administration of the anorectic hormone leptin significantly decreases breakpoints for HFHS food in mice. These findings are consistent with previously reported actions of food deprivation and leptin on reward thresholds in rats12-14.
When using this protocol it is important to keep in mind that acquisition of food-maintained operant responding is significantly enhanced by regular training (every 1-2 days) and by increasing the motivational state of the mouse by implementing a food restriction regimen. In addition, ensuring that mice exhibit relatively stable responding on one reinforcement schedule before moving on to more complex schedules (e.g, FR1 to FR5) is key to minimizing variable performance during testing. Despite the application of these steps, it is not unusual to obtain some variability especially between different mice. For this reason, it is highly recommended to use a within-subjects (repeated measures) design when possible to remove across subject variability. Although within-subject testing is not possible for investigating stable genetic modifications (i.e., knockout versus control mice) it can be used to examine the influence of drugs, lesions or acute genetic (e.g., optogenetic) manipulations.
Obesity and obesity-related illnesses are a growing problem that has increased the urgency to identify the molecules and neural pathways involved. Accumulating evidence highlights the important role of brain reward circuitry in food craving, increased caloric intake and weight gain. The PR task is a valuable tool for measuring the impact of neurobiological, genetic or pharmacological manipulations on the rewarding effects of food.

<table><tbody><tr><td>Mouse operant chambers</td><td><a href="http://www.med-associates.com/" target="_blank"><img src="/img/new_window.png"> Med Associates, Inc.</a></td><td>ENV-009A</td><td>Med Associates Inc.&lt;br/&gt;PO Box 319 &lt;br/&gt; St. Albans, Vermont 05478, &lt;br/&gt; USA &lt;br/&gt; Phone: (802) 527-2343</td></tr><tr><td>8 inputs/16 inputs Smart Control Connection Panel</td><td><a href="http://www.med-associates.com/" target="_blank"><img src="/img/new_window.png"> Med Associates, Inc.</a></td><td>SG-716B</td><td></td></tr><tr><td>Ultra-light retractable mouse lever</td><td><a href="http://www.med-associates.com/" target="_blank"><img src="/img/new_window.png"> Med Associates, Inc.</a></td><td>ENV-312</td><td></td></tr><tr><td>Pellet Dispenser</td><td><a href="http://www.med-associates.com/" target="_blank"><img src="/img/new_window.png"> Med Associates, Inc.</a></td><td>ENV-203</td><td></td></tr><tr><td>House Light</td><td><a href="http://www.med-associates.com/" target="_blank"><img src="/img/new_window.png"> Med Associates, Inc.</a></td><td>ENV-215</td><td></td></tr><tr><td>Stimulus Light</td><td><a href="http://www.med-associates.com/" target="_blank"><img src="/img/new_window.png"> Med Associates, Inc.</a></td><td>ENV-221</td><td></td></tr><tr><td>Dust-free Precision 20 mg high-fat &amp;amp; high-sugar pellets</td><td>Bio-Serv</td><td>F-06649</td><td>Bio-Serv &lt;br/&gt; One 8th Street, Suite 1 &lt;br/&gt;Frenchtown, NJ 08825, USA &lt;br/&gt; Phone: (800)-996-9908</td></tr><tr><td colspan="4">&lt;strong&gt;Table 1.&lt;/strong&gt; Specific equipment and material</td></tr></tbody></table>

progressive-ratio responding for palatable high-fat and high-sugar food in mice

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

The aim of the following experiment is to train and test mice in a food motivated task using a progressive ratio schedule of reinforcement. This is achieved by first food restricting the mice to 60 to 70%of Ad Liam food intake to a stable reduction of five to 10%in body weight. To enhance the acquisition of food motivated lever pressing behavior, the mice are then trained to lever press for a high fat, high sugar food reward in the fixed ratio one, using an auto shaping procedure.Miser then trained to respond for food on more complex schedules of reinforcement, including fixed ratio five, and then finally on the progressive ratio task, which provides a reliable measure of the rewarding effects of food. Next, after stable performance on the progressive ratio schedule, these mice are either food deprived for 12 to 24 hours or given intraperitoneal injection of retic agents to validate alterations in the food motivated behavior on the progressive ratio schedule of reinforcement. The results of this experiment show that food deprivation increases the food motivated behavior, whereas administration of the retic hormone leptin decreases the food motivated behavior for high fat and high sugar food rewards.Upper conditioning tasks provide a useful means to evaluate the rewarding and motivational effects of different stimuli and behavior. The progressive ratio task is a well-validated schedule of reinforcement that has widely been used to assess the rewarding effect of drug of abuse and natural rewards such as food, although more commonly used in red studies application of the progressive ratio opera tasks to mice has become increasingly important with the growing generation of genetically engineered mouse models. In particular, the use of mouse models in obesity research and the recognition that the rewarding effects of palatable calorically rich food significantly contribute to overeating and obesity has emphasized the need to measure food reward in mice.Here we explain and discuss the methods used to train and test mice to liver press for a palatable high fat, high sugar food on a progressive ratio schedule of reinforcement Before beginning. The protocol ensure that all procedures involving the use of animals have been approved by the institutional animal care. In use committee, it is recommended to test in the dark phase of the light dark cycle if needed.Acclimate mice to a reverse light dark cycle for seven to 10 days. At this point, the mice are provided with ad libitum access to standard chow and water Following this 10 day acclimation period. The mice are singly housed and food restricted by providing 60 to 70%of ad libitum food intake until they reach 90 to 95%of their free feeding body weights.This facilitates acquisition of leave press responding weigh the mice daily. Upon reaching five to 10%weight loss, the daily food allotted is adjusted to stabilize the lower body weights for the remainder of the training period three days prior to the first training session, transport the mice in their home cages to the testing room, then remove each mouse from its cage one at a time, and handle the mouse for two to three minutes. After handling, return the mouse to the home cage, acclimate the mice to the room and handling for three consecutive days the day prior to the start of training.10 to 15 high fat high sugar pellets are placed into the home cage to prevent the potential influence of food. Neo phobia on operant performance, 20 milligrams of high fat, high sugar, dust-free precision food pellets containing 49%killer cow as fat are used here. Each mouse operant chamber is housed inside a sound attenuating cubicle with ventilating fan.The mouse operant chambers are equipped with two ultra light retractable levers positioned on either side of the food receptacle. A stimulus light is located above each lever and there is a house light at the top of the cage. Appellate dispenser is located outside of the chamber.The chambers are connected to a PC computer with Med PC four software via a smart control interface cabinet. Mice are introduced to the operant chamber. The initial training paradigm consists of a fixed ratio reinforcement schedule, one whereby a single lever press elicits the delivery of a food pellet into the receptacle.Only one lever is designated as active and triggers the delivery of a food reward. The allocation of right and left levers is counterbalanced between mice. Training requires an also shaping procedure whereby crushed food pellets are placed on the lever to enhance lever pressing behavior.During this initial training, an active lever response triggers delivery of the food reward and there is no delay between lever pressing and food delivery. Once mice have learned to press the lever a five second time, timeout is initiated during which additional leave of pressing does not result in the delivery of a pellet. The timeout period allows for mice to consume the pellet.Longer timeout periods may be used if necessary after the timeout period pressing the active lever. Once again, results in the release of a food pellet each fixed ratio one five seconds. Timeout training session last one hour or when 50 pellets have been delivered.Opera and training continues for seven to 10 days until acquisition of food maintained opera and responding mice exhibiting discrimination of equal to or greater than three to one for the active versus inactive lever and obtaining equal to or greater than 20 rewards per session. Over three consecutive sessions are considered to have achieved acquisition criteria. Mice that do not reach acquisition criteria by the seven to 10 day period undergo further training for an additional five to seven days.Mice are excluded from testing when there is no progress by the fifth training day. It is important to keep in mind that impaired operant acquisition may be an outcome of certain pharmacological and genetic interventions and should therefore be documented. Following three successive sessions of obtaining equal to or greater than 20 pellets, the schedule has increased to the FR five T oh five seconds protocol in which five active leave presses triggers the delivery of the food pellet.Training on the FR five T oh five seconds schedule last about three to five days until responding is stable. The meister then trained in the progressive ratio schedule of reinforcement. The response ratio schedule during PR testing can be calculated as per Richardson and Roberts, where R is equal to the number of food rewards already earned, plus one.Thus, the number of responses required to earn a food reward follow the order, 1, 2, 4 6 9 12 15, 20, 25, 32, 40 50, 62, 77, 95, and so on. The final ratio completed is the break point. The PR session lasts a maximum of one hour failure to press the lever in any 10 minute period.Results in termination of the session. Performance on the PR schedule of reinforcement is considered stable when the number of rewards earned in a one hour session deviates by no more than 20%for at least three consecutive days to validate food maintained operant responding in the PR task, breakpoint responding following manipulations known to modulate food reward are assessed. A relatively easy test to carry out is food deprivation, which will increase the motivational state of the mouse for food and thereby increase break points.Mice are first tested in the PR task during a period of ad liberton feeding until stable baseline performance is achieved the day after the last baseline day. Food is removed from the home cage and mice are then tested in the PR task 12 to 24 hours after the start of the fast, or to investigate an opposite effect on breakpoint thresholds, one can test peripheral administration of the antic hormone. Leptin PR testing is carried out following an IP injection of PBS on one day and then after injection of five milligrams per kilogram of leptin on the following day.Leptin is injected one hour prior to PR testing as seen here. Fasting increases the rewarding effects of food a 24 hour fast, significantly increased break points on a progressive ratio schedule of reinforcement in mice as seen here. Leptin decreases food reward in mice since peripheral leptin administration decreased breakpoint response thresholds While attempting this procedure, it's important to remember that acquisition of food maintain operant responding is significantly enhanced by regular training and by increasing the motivational state of the mouse by implementing a food restriction regimen.In addition, ensuring that mice exhibit relatively stable responding on one reinforcement schedule before moving on to more complex schedules is key to minimizing variable performance during testing. Obesity and obesity related illnesses are a growing problem that has increased the urgency to identify the molecules and the neural pathways involved. The progressive ratio schedule is a valuable tool for measuring the impact of neurobiological, genetic, or pharmacological manipulations on the rewarding effects of the food.

progressive-ratio-responding-for-palatable-high-fat-high-sugar-food

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Method to assess the impact of training on motor skills is a useful tool. Unfortunately, most behavioral assessments can be labor intensive and/or expensive.We describe here a robotic method of assessing prehension (reach-to-grasp) skill in mice.

Evaluating Skilled Prehension in Mice Using an Auto-Trainer

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice.

Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System

A binge-eating model was established in mice using intermittent access to M&amp;M's (a highly palatable food made of fat and sugar). Mice with intermittent access to this high-fat, high-sugar food showed stress-like behaviors, increased caloric intake, and a preference for that food over standard chow compared to mice with continuous or no access to the food.

Intermittent Binge-Intake Model in Mice

Learn progressive-ratio operant conditioning to measure food reward motivation in mice using high-fat, high-sugar pellets. This method validates how food deprivation and leptin alter motivated behavior.

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

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